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Lecture6 - MCB 110L Spring 2011 Lecture 6 Qing Zhong 306...

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MCB 110L Spring 2011 Lecture 6 2/4/2011 Qing Zhong 306 Barker Hall
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ATP Cin8 (MTs) ADP + Pi Malachite Green Enzyme Product Substrate 1:1 Enzyme activity (U), catalyze one micromole( μ mol) of substrate(ATP) per minute . We are measuring Pi, which is a product with 1:1 ratio to ATP. We are calculating Pi extinction coefficient, Pi μ mol, Pi μ mol/min, not about Cin8. More about enzyme activity International Unit : 1IU of Insulin is equal to the amount required to reduce the concentration of blood glucose in a fasting rabbit to 45 mg/dl (2.5 mmol/L).
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Malachite green H 3 PO 4 (Pi) + 12 H 2 MoO 4 = PO 4 (MoO 3 ) 12 3- + 12 H 2 O + 3H + molybdate Phosphomolybdate complex PO 4 (MoO 3 ) 12 3- + Malachite green = Green Citrate quenches the reaction by binding to free molybdate . 12 H 2 MoO 4 + Malachite green = yellow
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Enzyme activity is about “speed” • How many molecules of substrates can one molecule of enzyme catalyze per minutes? Substrates μ mol/min II: μ mol of enzyme (turnover rate) -best description of enzyme activity I: mg of enzyme -specific activity (impure sample) III: volume of enzyme (ml) -non-standard way -OK for a fixed concentration Otherwise need to convert to μ mol or mg
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•You add 10 μL of a diluted enzyme stock (100X dilution, stock 0.5mg/ml) to a 175 uL reaction (10ul included).
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